Electroporation is a technique wherein cells are stimulated to take up material from their surrounding medium by placing the medium containing the material and a suspension of cells between two electrodes and exposing the cells to an electrical impulse (Wong, T. K., et al. (1982) Biochem and Biophys. Research Commun. 107, 584-587); Neumann, E., et al. (1982) Eur. Mol. Biol. Org. 1, 841-845; Potter, H., et al. (1984), Proc. Natl. Acad. Sci 81, 7161-7165). This electrical impulse is most often generated by applying a pulsed direct voltage to the electrodes to produce a pulsed direct current between the two electrodes. A suggested mechanism for this electroporation phenomenon involves the formation of transient holes or pores in the cell membrane through which the surrounding medium containing the material may enter the cell. This is followed by reclosure of the pores and continuation of normal cell growth for those cells which survive the electroporation. This electroporation technique has been most widely applied to the introduction of foreign DNA into cells.
Electroporation of DNA is one of several methods used to introduce DNA into cells (commonly referred to as transfection). Examples of other transfection methods include calcium phosphate co-precipitation (Graham, F. L., et al. (1973), Virology 52, 456-467), diethylaminoethyl-Dextran treatment (McCutchan, J. H., et al. (1968), J. Natl. Canc. Ins. 41. 351-357), protoplasfusion (Schaffner, W. (1980), Proc. Natl. Acad. Sci. 77, 2163-2167), microinjection (Capecchi, M. R. (1980), Cell 22, 479-488) and retrovirus infection (Weiss, R. N., et al. (1982) eds. "RNA Tumor Viruses", Cold Spring Harbor Laboratory, New York).
A common and significant problem with each of these transfection techniques is the low nonreproducible transfection frequency associated with these methods. Moreover, transfection by the calcium phosphate co-precipitation method or by the electroporation of a cell suspension is often not possible since some cell types are killed, or have very low viability when so treated. In addition, some cell types cannot be transfected by the calcium phosphate co-precipitation method or by suspension electroporation, or can only be transfected at extremely low frequencies by these methods.
A significant additional problem is encountered when the suspension electroporation technique is used to transfect cell types which may be grown only when attached to the surface of a culture vessel or to other cells. Prior to electroporation, such cells are treated, either with a proteolytic enzyme such as trypsin or with a chelating agent such as ethylenediaminetetraacetic acid (EDTA), to remove the cells from the culture vessel surface, and from each other, to form a suspension of such cells. The cell suspension together with the DNA to be transfected is transferred to an electroporation device containing two electrodes. A direct electric current is then generated between the electrodes. The cell suspension is then returned to a culture vessel containing selection medium capable of differentiating transfected cells from unsuccessful transformants and dead cells. Such methodology and apparatus are reported by Wong and Neumann (Wong, T. K., et al. (1982) Biochem and Biophys. Research Commun. 107, 584-587); Neumann, E., et al. (1982) Eur. Mol. Biol. Org. 1, 841-845). As a consequence of such enzymatic or chelation treatment and manipulation, cell viability and transfection frequency are markedly decreased.
The design of these electroporation devices severely restricts their utility. It is not clear whether pore formation results from an interaction between the electric field and induced dipoles in the plasma membrane (Neumann, E., et al. (1982) Eur. Mol. Biol. Org. 1, 841-845), a current generated through the cells, transient heat generated by the electrical power through the cells, or a combination of the above effects. Regardless of the mechanism involved, electroporation typically requires the application of about 0.5-10 kilovolts per centimeter between two electrodes to induce transfection at reasonably high frequencies. Thus, the two electrodes have been placed in close proximity to each other, e.g., 2-5 millimeters for use with applied voltages ranging from about 500-5000 volts. Any increase in electrode separation requires a conconmittant increase in applied voltage and has therefore been limited by the availability of power sources capable of delivering such pulsed voltages and the adverse effects of such high voltages on cell viability and transfection frequency. Since transfection presumably occurs between the two electrodes, the electroporation devices heretofore used for transfection have been limited to a relatively small volume wherein the electroporation occurs. Moreover, such electroporation devices have not been adaptable to the electroporation of attachment-dependent cells in situ.
Accordingly, it is an object herein to provide convenient and reproducible electroporation processes for the transfection of cell suspensions and cells adhered to a surface resulting in increased transfection frequencies, including effective transformation of heretofore nontransfectable cell types, and increased viability of transfected cells.
A further object of the invention is to provide electroporation apparatus for transfecting cell suspensions and attachment-dependent cell types in situ without the need for removing such cells from their growth surface.